Pulsed infrared excitation was used to induce autofluorescence of the energy-carrying molecules NADH and NADPH in living brain slices from mouse, cultured neonatal rat hippocampal and cortical neurons, and cultured PC12 cells. Levels of (putatively) NADH and NADPH could be increased by bath application of NaCN and decreased by bath application of mitochondrial proton-gradient uncouplers. Spectra of the autofluorescence closely match the solution spectra of NAD(P)H. Diffuse autofluorescence was observed in some sections of the mouse brain, while consistently bright autofluorescence was observed in an unidentified, possibly epithelial, cell-type lining capillaries. Identification of individual neurons or processes in a brain slice was difficult. However, autofluorescence imaging of NAD(P)H promises to be a useful non-invasive indicator of cellular metabolism in neural systems.